RESUMEN
BACKGROUND: Partial negative allosteric modulators (NAM) of the metabotropic glutamate 5 (mGlu5) receptor are an excellent alternative to full antagonists and NAMs because they retain therapeutic effects and have a much broader therapeutic window. Here, we investigated whether partial mGlu5 NAM, 2-(2-(3-methoxyphenyl)ethynyl)-5-methylpyridine (M-5MPEP), induced a fast and sustained antidepressant-like effect, characteristic of rapid-acting antidepressant drugs (RAADs) like ketamine, in mice. METHODS: A tail suspension test (TST) was used to investigate acute antidepressant-like effects. Sustained effects were studied 24 h after the four intraperitoneal (ip) administrations using the splash test, designed to measure apathy-like state, the sucrose preference test (SPT), reflecting anhedonia, and the TST. Western blot and ELISA techniques were used to measure brain-derived neurotrophic factor (BDNF) and selected protein levels. METHODS: A tail suspension test (TST) was used to investigate acute antidepressant-like effects. Sustained effects were studied 24 h after the four intraperitoneal (ip) administrations using the splash test, designed to measure apathy-like state, the sucrose preference test (SPT), reflecting anhedonia, and the TST. Western blot and ELISA techniques were used to measure brain-derived neurotrophic factor (BDNF) and selected protein levels. CONCLUSION: Partial mGlu5 receptor NAM, M-5MPEP, induced rapid and sustained antidepressant-like effects in the BDNF-dependent mechanism and enhanced (R)-ketamine action in mice, indicating both substances' convergent mechanisms of action and the possibility of their practical use in treating depression as RAAD.
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Antidepresivos , Factor Neurotrófico Derivado del Encéfalo , Suspensión Trasera , Ketamina , Piridinas , Receptor del Glutamato Metabotropico 5 , Animales , Masculino , Ratones , Regulación Alostérica/efectos de los fármacos , Anhedonia/efectos de los fármacos , Antidepresivos/farmacología , Conducta Animal/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Depresión/tratamiento farmacológico , Modelos Animales de Enfermedad , Ketamina/farmacología , Ketamina/administración & dosificación , Piridinas/farmacología , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidoresRESUMEN
Background: Altered sensory processing is a pervasive symptom in individuals with Autism Spectrum Disorders (ASD); people with Phelan McDermid syndrome (PMS), in particular, show reduced responses to sensory stimuli. PMS is caused by deletions of the terminal end of chromosome 22 or point mutations in Shank3. People with PMS can present with an array of symptoms including ASD, epilepsy, gastrointestinal distress, and reduced responses to sensory stimuli. People with PMS are often medicated to manage behaviors like aggression and/or self-harm and/or epilepsy, and it remains unclear how these medications might impact perception/sensory processing. Here we test this using zebrafish mutant shank3ab PMS models that likewise show reduced sensory responses in a visual motor response (VMR) assay, in which increased locomotion is triggered by light to dark transitions. Methods: We screened three medications, risperidone, lithium chloride (LiCl), and carbamazepine (CBZ), prescribed to people with PMS and one drug, 2-methyl-6-(phenylethynyl) pyridine (MPEP) tested in rodent models of PMS, for their effects on a sensory-induced behavior in two zebrafish PMS models with frameshift mutations in either the N- or C- termini. To test how pharmacological treatments affect the VMR, we exposed larvae to selected drugs for 24 hours and then quantified their locomotion during four ten-minute cycles of lights on-to-off stimuli. Results: We found that risperidone normalized the VMR in shank3 models. LiCl and CBZ had no effect on the VMR in any of the three genotypes. MPEP reduced the VMR in wildtype (WT) to levels seen in shank3 models but caused no changes in either shank3 model. Finally, shank3 mutants showed resistance to the seizure-inducing drug pentylenetetrazol (PTZ), at a dosage that results in hyperactive swimming in WT zebrafish. Conclusions: Our work shows that the effects of drugs on sensory processing are varied in ways that can be highly genotype- and drug-dependent.
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Trastornos de los Cromosomas , Percepción , Pez Cebra , Animales , Humanos , Cromosomas Humanos Par 22 , Proteínas del Tejido Nervioso/genética , Risperidona/farmacología , Pez Cebra/genética , Trastornos de los Cromosomas/tratamiento farmacológico , Trastornos de los Cromosomas/genética , Modelos Animales de Enfermedad , Cloruro de Litio/farmacología , Carbamazepina/farmacología , Percepción/efectos de los fármacosRESUMEN
The blockade of metabotropic glutamate receptor type 5 (mGluR5) was previously found to reduce fat accumulation in HEPG2 cells. Here, we evaluated the effects of mGluR5 blockade in a mouse model of steatosis. Male ob/ob mice fed a high-fat diet were treated with MPEP or vehicle. After 7 weeks, liver biopsies were collected, and nuclei were isolated from fresh tissue. Lipid droplet area and collagen deposition were evaluated on tissue slices; total lipids, lipid peroxidation, and ROS were evaluated on tissue homogenates; PPARα, SREBP-1, mTOR, and NF-κB were assayed on isolated nuclei by Western Blot. Target genes of the above-mentioned factors were assayed by RT-PCR. Reduced steatosis and hepatocyte ballooning were observed in the MPEP group with respect to the vehicle group. Concomitantly, increased nuclear PPARα and reduced nuclear SREBP-1 levels were observed in the MPEP group. Similar trends were obtained in target genes of PPARα and SREBP-1, Acox1 and Acc1, respectively. MPEP administration also reduced oxidative stress and NF-κB activation, probably via NF-κB inhibition. Levels of common markers of inflammation (Il-6, Il1ß and Tnf-α) and oxidative stress (Nrf2) were significantly reduced. mTOR, as well as collagen deposition, were unchanged. Concluding, MPEP, a selective mGluR5 negative allosteric modulator, reduces both fat accumulation and oxidative stress in a 7-week murine model of steatosis. Although underlying mechanisms need to be further investigated, this is the first in vivo study showing the beneficial effects of MPEP in a murine model of steatosis.
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Hígado Graso , Enfermedad del Hígado Graso no Alcohólico , Ratones , Masculino , Animales , Hígado/patología , Ratones Obesos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , FN-kappa B/farmacología , PPAR alfa , Modelos Animales de Enfermedad , Hígado Graso/tratamiento farmacológico , Hígado Graso/genética , Hígado Graso/patología , Dieta Alta en Grasa/efectos adversos , Serina-Treonina Quinasas TOR , Enfermedad del Hígado Graso no Alcohólico/patología , Ratones Endogámicos C57BLRESUMEN
RATIONALE AND OBJECTIVE: Post-traumatic stress disorder (PTSD) is a prevalent and debilitating psychiatric disorder. However, its specific etiological mechanism remains unclear. Previous studies have shown that traumatic stress changes metabotropic glutamate receptor 5 (mGluR5) expression in the hippocampus (HIP) and prefrontal cortex (PFC). More importantly, mGluR5 expression is often accompanied by alterations in brain-derived neurotrophic factor (BDNF). Furthermore, BDNF/tropomyosin-associated kinase B (TrkB) signaling plays multiple roles, including roles in neuroplasticity and antidepressant activity, by regulating glutamate transporter-1 (GLT-1) expression. This study aims to explore the effects of inhibiting mGluR5 on PTSD-like behaviors and BDNF, TrkB, and GLT-1 expression in the HIP and PFC of inevitable foot shock (IFS)-treated rats. METHODS: Seven-day IFS was used to establish a PTSD rat model, and 2-methyl-6-(phenylethynyl)-pyridine (MPEP) (10 mg/kg, intraperitoneal injection) was used to inhibit the activity of mGluR5 during IFS in rats. After modeling, behavioral changes and mGluR5, BDNF, TrkB, and GLT-1 expression in the PFC and HIP were examined. RESULTS: First, the IFS procedure induced PTSD-like behavior. Second, IFS increased the expression of mGluR5 and decreased BDNF, TrkB, and GLT-1 expression in the PFC and HIP. Third, the mGluR5 antagonist blocked the above behavioral and molecular alterations. CONCLUSIONS: mGluR5 was involved in IFS-induced PTSD-like behavior by changing BDNF, TrkB, and GLT-1 expression.
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Factor Neurotrófico Derivado del Encéfalo , Transportador 2 de Aminoácidos Excitadores , Receptor del Glutamato Metabotropico 5 , Receptor trkB , Trastornos por Estrés Postraumático , Animales , Ratas , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipocampo/metabolismo , Corteza Prefrontal/metabolismo , Receptor del Glutamato Metabotropico 5/genética , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Trastornos por Estrés Postraumático/genética , Trastornos por Estrés Postraumático/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismoRESUMEN
BACKGROUND: The metabotropic glutamate receptor 5 (mGluR5) is widely expressed in postsynaptic neurons and plays a vital role in the synaptic plasticity of the central nervous system. mGluR5 is a coreceptor for amyloid-ß (Aß) oligomer, and downregulation or pharmacological blockade of mGluR5 presents the therapeutic potential of Alzheimer's disease (AD). However, the abnormality of mGluR5 in the pathogenesis of AD and its mechanism of pathology is not clear. OBJECTIVE: In this study, we would like to investigate the expression of mGluR5 in the process of AD and explore the effects and the underlying mechanisms of antagonizing mGluR5 on cognitive function, synaptic structure, and inflammation in 5xFAD mice. METHODS: mGluR5 expression and interactions with PrPc in 5XFAD mice were detected using western blot and co-immunoprecipitation. The selective mGluR5 antagonist MPEP was infused into 4-month-old 5XFAD mice for 60 consecutive days. Then, cognitive function, AD-like pathology and synaptic structure were measured. Further observations were made in mGluR5 knockdown 5XFAD mice. RESULTS: mGluR5 expression was increased with Aß levels at 6 months in 5XFAD mice. mGluR5 antagonist rescued cognitive disorders, promoted synaptic recovery, and alleviated both the Aß plaque load and abnormal hyperphosphorylation in 6-month-old 5XFAD mice. Meanwhile, the results were validated in mGluR5 knockdown mice. Blockade of mGluR5 efficiently alleviates AD-like pathologies by inhibiting the PI3K/AKT/mTOR pathway and activates autophagy in 5XFAD mice. Furthermore, antagonism of mGluR5 attenuated neuroinflammation by inactivating the IKK/NF-κB pathway. CONCLUSION: These findings suggest that mGluR5 may be an effective drug target for AD treatment, and inhibition of the mGluR5/PI3K-AKT pathway alleviates AD-like pathology by activating autophagy and anti-neuroinflammation in 5XFAD mice.
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Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Ratones Transgénicos , Receptor del Glutamato Metabotropico 5/metabolismo , Péptidos beta-Amiloides/metabolismo , Autofagia , Modelos Animales de EnfermedadRESUMEN
In this study, we investigated the cross-talk between mGlu1 and CB1 receptors in modulating GABA hippocampal output in whole-cell voltage clamp recordings in rat hippocampal acute slices, in organotypic hippocampal slices exposed to oxygen and glucose deprivation (OGD) and in gerbils subjected to global ischemia. CB1 receptor expression was studied using immunohistochemistry and the CA1 contents of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by LC-MS/MS. Our results show that mGlu1 receptor antagonists enhance sIPSCs in CA1 pyramidal cells and the basal and ischemic hippocampal release of GABA in vivo in a manner that is mediated by CB1 receptor activation. In hippocampal slices exposed to OGD and in ischemic gerbils, mGlu1 receptor antagonists protected CA1 pyramidal cells against post-ischemic injury and this effect was reduced by CB1 receptor activation. OGD induced a transient increase in the hippocampal content of AEA and this effect is prevented by mGlu1 receptor antagonist. Finally, OGD induced a late disruption of CB1 receptors in the CA1 region and the effect was prevented when CA1 pyramidal cells were protected by mGlu1 antagonists. Altogether, these results suggest a cooperative interaction between mGlu1 receptors and the endocannabinoid system in the mechanisms that lead to post-ischemic neuronal death.
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Endocannabinoides , Fármacos Neuroprotectores , Animales , Cromatografía Liquida , Endocannabinoides/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Gerbillinae/metabolismo , Glucosa/farmacología , Fármacos Neuroprotectores/farmacología , Oxígeno/farmacología , Ratas , Receptor Cannabinoide CB1 , Receptores Presinapticos , Transmisión Sináptica/fisiología , Espectrometría de Masas en Tándem , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Proinflammatory markers were found in brains of Parkinson's disease (PD) patients. After years of L-Dopa symptomatic treatment, most PD patients develop dyskinesias. The relationship between inflammation and L-Dopa-induced dyskinesias (LID) is still unclear. We previously reported that MPEP (a metabotropic glutamate receptor 5 antagonist) reduced the development of LID in de novo MPTP-lesioned monkeys. We thus investigated if MPEP reduced the brain inflammatory response in these MPTP-lesioned monkeys and the relationship to LID. The panmacrophage/microglia marker Iba1, the phagocytosis-related receptor CD68, and the astroglial protein GFAP were measured by Western blots. The L-Dopa-treated dyskinetic MPTP monkeys had increased Iba1 content in the putamen, substantia nigra, and globus pallidus, which was prevented by MPEP cotreatment; similar findings were observed for CD68 contents in the putamen and globus pallidus. There was a strong positive correlation between dyskinesia scores and microglial markers in these regions. GFAP contents were elevated in MPTP + L-Dopa-treated monkeys among these brain regions and prevented by MPEP in the putamen and subthalamic nucleus. In conclusion, these results showed increased inflammatory markers in the basal ganglia associated with LID and revealed that MPEP inhibition of glutamate activity reduced LID and levels of inflammatory markers.
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Discinesia Inducida por Medicamentos , Enfermedad de Parkinson , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/efectos adversos , Animales , Encéfalo/metabolismo , Humanos , Inflamación/metabolismo , Levodopa/metabolismo , Macaca fascicularis , Enfermedad de Parkinson/metabolismo , Piridinas/farmacología , Receptor del Glutamato Metabotropico 5/metabolismoRESUMEN
We know surprisingly little about the sex differences in the neurobiology of cocaine addiction, except females are more susceptible to the rewarding effects of cocaine than their male counterparts. Only a handful of recent studies have examined the neurobiology of cocaine-induced conditioned place preference (CPP) memory among female rodents. We contribute to this emerging line of research by documenting sex differences in cocaine-associated memory and illustrating the underlying signaling pathways in five experiments. Rimonabant (Rim), a cannabinoid CB1 antagonist and inverse agonist, exerted a facilitating effect for low-dose cocaine and an impairing effect for high-dose cocaine CPP memory in male mice, as in our previous study, but not in female mice. Nor did we observe the effect exist among CB1 knockout male mice, which indicated that the CB1 receptors played a mediating role. We also found that the metabotropic glutamate receptor 5 (mGluR5) was located in the same signaling pathway as CB1 in male mice. To clarify the mechanisms behind the sex differences, we used ovariectomized (OVX) female mice with estradiol benzoate (EB) replacement. In the OVX female mice, we showed that Rim-alone and EB-alone, but not Rim-and-EB-combined, facilitated the low-dose cocaine CPP memory. Moreover, 4-hydroxytamoxifen (4-OHT), an estrogen receptor (ER) antagonist, blocked Rim's and EB's facilitating effect. Finally, 2-methyl-6-(phenylethynyl)pyridine (MPEP), an mGluR5 antagonist, partially blocked EB's facilitating effect. In sum, we identified sex-specific effects of Rim on cocaine-induced CPP memory and the respective signaling pathways: mGluR5-CB1 for male mice and ER-mGluR5-CB1 for female mice. These findings may have merits for the development of sex-specific treatment for cocaine addiction.
Asunto(s)
Cocaína/farmacología , Receptor Cannabinoide CB1 , Rimonabant/farmacología , Animales , Antagonistas de Receptores de Cannabinoides , Trastornos Relacionados con Cocaína , Estradiol , Femenino , Masculino , Ratones , Receptor del Glutamato Metabotropico 5 , Caracteres SexualesRESUMEN
We previously demonstrated that the blockade of mGluR5 by 2-methyl-6(phenylethynyl)pyridine (MPEP) reduces both cold and warm ischemia/reperfusion injury. Here we evaluated whether MPEP reduces the hepatic preservation injury in rat livers from cardiac-death-donors (DCDs). Livers from DCD rats were isolated after an in situ warm ischemia (30 min) and preserved for 22 h at 4 °C with UW solution. Next, 10 mg/Kg MPEP or vehicle were administered 30 min before the portal clamping and added to the UW solution (3 µM). LDH released during washout was quantified. Liver samples were collected for iNOS, eNOS, NO, TNF-α, ICAM-1, caspase-3 and caspase-9 protein expression and nuclear factor-erythroid-2-related factor-2 (Nrf2) gene analysis. Lower LDH levels were detected in control grafts versus DCD groups. An increase in eNOS and NO content occurred after MPEP treatment; iNOS and TNF-α content was unchanged. ICAM-1 expression was reduced in the MPEP-treated livers as well as the levels of caspase-3 and caspase-9. Nrf2, oxidative stress-sensitive gene, was recovered to control value by MPEP. These results suggest that MPEP can be used to reclaim DCD livers subjected to an additional period of cold ischemia during hypothermic storage. MPEP protects against apoptosis and increased eNOS, whose overexpression has been previously demonstrated to be protective in hepatic ischemia/reperfusion damage.
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Isquemia Fría/efectos adversos , Muerte , Hígado/metabolismo , Preservación de Órganos/métodos , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Daño por Reperfusión/prevención & control , Isquemia Tibia/efectos adversos , Animales , Trasplante de Hígado , Masculino , Ratas , Ratas Wistar , Daño por Reperfusión/etiología , Daño por Reperfusión/patología , Donantes de TejidosRESUMEN
Mass spectrometry (MS) binding assays are a label-free alternative to radioligand or fluorescence binding assays, so the readout is based on direct mass spectrometric detection of the test ligand. The study presented here describes the development and validation of a highly sensitive, rapid, and robust MS binding assay for the quantification of the binding of the metabotropic glutamate 5 (mGlu5) negative allosteric modulator (NAM), MPEP (2-methyl-6-phenylethynylpyridine) at the mGlu5 allosteric binding site. The LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometric) analytical method was established and validated with a deuterated analogue of MPEP as an internal standard. The developed MS binding assay described here allowed for the determination of MS binding affinity estimates that were in agreement with affinity estimates obtained from a tritiated MPEP radioligand saturation binding assay, indicating the suitability of this methodology for determining affinity estimates for compounds that target mGlu5 allosteric binding sites. Graphical abstract.
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Cromatografía Líquida de Alta Presión/métodos , Receptor del Glutamato Metabotropico 5/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Sitio Alostérico , Células HEK293 , Humanos , Ligandos , Límite de Detección , Unión Proteica , Ensayo de Unión Radioligante , Reproducibilidad de los ResultadosRESUMEN
As part of the G-protein coupled receptor (GPCR) family, metabotropic glutamate (mGlu) receptors play an important role as drug targets of cognitive diseases. Selective allosteric modulators of mGlu subtype 5 (mGlu5) have the potential to alleviate symptoms of numerous central nervous system disorders such as schizophrenia in a more targeted fashion. Multiple mGlu5 positive allosteric modulators (PAMs), such as 1-(3-fluorophenyl)-N-((3-fluorophenyl)-methylideneamino)-methanimine (DFB), 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide (CDPPB), and 4-nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide (VU-29), exert their actions by binding to a defined allosteric site on mGlu5 located in the seven-transmembrane domain (7TM) and shared by mGlu5 negative allosteric modulator (NAM) 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Actions of the PAM N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) are mediated by a distinct allosteric site in the 7TM domain different from the MPEP binding site. Experimental evidence confirms these findings through mutagenesis experiments involving residues F585 (TM1) and A809 (TM7). In an effort to investigate mGlu5 PAM selectivity for this alternative allosteric site distinct from MPEP binding, we employed in silico quantitative structure-activity relationship (QSAR) modeling. Subsequent ligand-based virtual screening prioritized a set of 63 candidate compounds predicted from a library of over 4 million commercially available compounds to bind exclusively to this novel site. Experimental validation verified the biological activity for seven of 63 selected candidates. Further, medicinal chemistry optimizations based on these molecules revealed compound VU6003586 with an experimentally validated potency of 174 nM. Radioligand binding experiments showed only partial inhibition at very high concentrations, most likely indicative of binding at a non-MPEP site. Selective positive allosteric modulators for mGlu5 have the potential for tremendous impact concerning devastating neurological disorders such as schizophrenia and Huntington's disease. These identified and validated novel selective compounds can serve as starting points for more specifically tailored lead and probe molecules and thus help the development of potential therapeutic agents with reduced adverse effects.
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Descubrimiento de Drogas/métodos , Receptor del Glutamato Metabotropico 5/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Animales , Ensayos Analíticos de Alto Rendimiento , Humanos , Receptor del Glutamato Metabotropico 5/química , Receptor del Glutamato Metabotropico 5/metabolismo , Relación Estructura-ActividadRESUMEN
Levodopa remains to be the mainstay for treatment of Parkinson disease (PD). Long-term levodopa treatment bears a risk for developing levodopa-induced dyskinesia (LID). LID significantly overshadows patients' quality of life and therapeutic efficacy of levodopa. Pre- and post-synaptic changes in dopamine secretion and signaling, along with altered glutamate receptor expression and glutamatergic signaling in striatal neurons, and the resulting disinhibition-like changes in the corticostriatal circuitry, lead to aberrant activity of motor cortex and formation of LID. Research has highlighted the role of group I metabotropic glutamate receptors especially the metabotropic glutamate receptor 5 (mGlu5) in formation of LID through potentiating of ionotropic glutamate NMDA receptors and dopamine D1/D5 receptors in direct pathway. Accordingly, MTEP and MPEP were the first mGlu5 receptor antagonists which were shown to attenuate LID in animal models through suppression of downstream signaling cascades involving mitogen-activated protein kinase (MAPK) and FosB/delta FosB activation, as well as modulation of prodynorphinegic, preproenkephalinergic, and GABA-ergic neurotransmission systems. Beneficial effects of other mGlu5 receptor antagonists such as AFQ056/mavoglurant and ADX48621/dipraglurant in amelioration of LID has been shown not only in animal models but also in clinical trials. Considering the presence of mGlu receptor dysregulation in rapid eye movement (REM) sleep behavior disorder and depression, which are prodromal signs of PD, along with the neuroprotective effects of mGlu receptor antagonists, and their cognitive benefits, potential effectiveness of mGlu receptor antagonists in early prevention of PD remains to be investigated.
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Discinesia Inducida por Medicamentos/tratamiento farmacológico , Discinesia Inducida por Medicamentos/fisiopatología , Levodopa/efectos adversos , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Animales , Discinesia Inducida por Medicamentos/metabolismo , Ácido Glutámico/metabolismo , Humanos , Receptores de Glutamato/metabolismoRESUMEN
Allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu5) have been proposed as potential therapies for various CNS disorders. These ligands bind to sites distinct from the orthosteric (or endogenous) ligand, often with improved subtype selectivity and spatio-temporal control over receptor responses. We recently revealed that mGlu5 allosteric agonists and positive allosteric modulators exhibit biased agonism and/or modulation. To establish whether negative allosteric modulators (NAMs) engender similar bias, we rigorously characterized the pharmacology of eight diverse mGlu5 NAMs. Radioligand inhibition binding studies revealed novel modes of interaction with mGlu5 for select NAMs, with biphasic or incomplete inhibition of the radiolabeled NAM, [3H]methoxy-PEPy. We assessed mGlu5-mediated intracellular Ca2+ (iCa2+) mobilization and inositol phosphate (IP1) accumulation in HEK293A cells stably expressing low levels of mGlu5 (HEK293A-rat mGlu5-low) and mouse embryonic cortical neurons. The apparent affinity of acetylenic NAMs, MPEP, MTEP and dipraglurant, was dependent on the signaling pathway measured, agonist used, and cell type (HEK293A-rat mGlu5-low versus mouse cortical neurons). In contrast, the acetylenic partial NAM, M-5MPEP, and structurally distinct NAMs (VU0366248, VU0366058, fenobam), had similar affinity estimates irrespective of the assay or cellular background. Biased modulation was evident for VU0366248 in mouse cortical neurons where it was a NAM for DHPG-mediated iCa2+ mobilization, but neutral with DHPG in IP1 accumulation assays. Overall, this study highlights the inherent complexity in mGlu5 NAM pharmacology that we hypothesize may influence interpretation when translating into preclinical models and beyond in the design and development of novel therapeutics for neuropsychiatric and neurological disorders.
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Regulación Alostérica/efectos de los fármacos , Ligandos , Receptor del Glutamato Metabotropico 5/metabolismo , Animales , Calcio/metabolismo , Sistema Nervioso Central , Femenino , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Cinética , Ratones , Embarazo , Cultivo Primario de Células , RatasRESUMEN
The fragile X mental retardation 1 knockout (Fmr1 KO) mouse replicates behavioral deficits associated with autism, fragile X syndrome, and schizophrenia. Less is known whether protein expression changes are consistent with findings in subjects with schizophrenia. In the current study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics to determine the protein expression of four subcellular fractions in the forebrains of Fmr1 KO mice vs. C57BL/6 J mice and the effect of a negative allosteric modulator of mGluR5-2-Methyl-6-(phenylethynyl)pyridine (MPEP)-on protein expression. Strain- and treatment-specific differential expression of proteins was observed, many of which have previously been observed in the brains of subjects with schizophrenia. Western blotting verified the direction and magnitude of change for several proteins in different subcellular fractions as follows: neurofilament light protein (NEFL) and 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) in the total homogenate; heterogeneous nuclear ribonucleoproteins C1/C2 (HNRNPC) and heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) in the nuclear fraction; excitatory amino acid transporter 2 (EAAT2) and ras-related protein rab 3a (RAB3A) in the synaptic fraction; and ras-related protein rab 35 (RAB35) and neuromodulin (GAP43) in the rough endoplasmic reticulum fraction. Individuals with FXS do not display symptoms of schizophrenia. However, the biomarkers that have been identified suggest that the Fmr1 KO model could potentially be useful in the study of schizophrenia.
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Antagonistas de Aminoácidos Excitadores/farmacología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Prosencéfalo/metabolismo , Proteoma/genética , Piridinas/farmacología , Esquizofrenia/genética , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/genética , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/metabolismo , Animales , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Eliminación de Gen , Ratones , Ratones Endogámicos C57BL , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Prosencéfalo/efectos de los fármacos , Proteoma/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Esquizofrenia/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Glioma is a primary brain tumor with high frequency and dismal prognosis. As there is no permanent cure available, identifying new therapy or mediator to augment the effectiveness of existing therapy is urgently needed. In the current study we tested the effect of group I metabotropic glutamate receptors (mGluRs): mGluR1 and mGluR5 on the viability of glioma cell lines. We analyzed cell viability using lactate dehydrogenase (LDH) release assay and evaluated apoptosis by propidium iodide (PI) staining. We used qPCR to evaluate change in mitochondrial gene expression and Western blot to evaluate the phosphorylation of Akt and ERK. Inhibition of mGluR5 by a selective antagonist MPEP under hypoxia promoted cell death, and induced expression of mitochondrial oxidative function related genes, with concurrent lowering of AKT phosphorylation level in glioma cell lines. Akt activation reversed mGluR5 inhibition on hypoxia-induced glioma cell death. These results suggest mGluR5 as a potential therapeutic target for hypoxic tumors such as malignant glioma.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Muerte Celular/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Glioma/metabolismo , Hipoxia/metabolismo , Piridinas/farmacología , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Glioma/patología , Humanos , Hipoxia/patología , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Levodopa (L-DOPA) is still the most effective drug for the treatment of Parkinson's disease (PD). However, the long-term therapy often triggers L-DOPA-induced dyskinesia (LID). Metabotropic glutamate receptor type 5 (mGluR5) is abundant in the basal ganglia, and its inhibition is thought to modulate postsynaptic excitatory synaptic transmission and glutamate hyperactivity in PD and LID. In this report, we examined the effects of mGluR5-specific antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) on LID and synaptic components in the PD model rat. We found the selective mGluR5 antagonist MPEP attenuated abnormal involuntary movements, prolonged the duration of rotational response, reversed the decrease of left forepaw adjusting steps, and reduced overexpression of striatal mGluR5 in the LID rats. Moreover, our results showed much thicker postsynaptic densities, narrower synapse cleft, as well as the increased ratio of perforated synapses induced by L-DOPA treatment, while coadministration of L-DOPA and MPEP reversed these postsynaptic effects. Finally, MPEP reduced overexpression of the two postsynaptic proteins (PSD-95 and SAP102) induced by L-DOPA treatment. Hence, these results provide evidence that aberrant neural plasticity at corticostriatal synapses in the striatum is closely correlated with the occurrence of LID, and targeted inhibition of mGluR5 by MPEP alleviates LID in the PD rat model.
Asunto(s)
Discinesia Inducida por Medicamentos/tratamiento farmacológico , Levodopa/efectos adversos , Oxidopamina/toxicidad , Piridinas/uso terapéutico , Sinapsis/metabolismo , Animales , Corteza Cerebral/patología , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large/metabolismo , Masculino , Neuropéptidos/metabolismo , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacosRESUMEN
Histaminergic (HA) neurons located in the tuberomamillary nucleus (TMN) of the posterior hypothalamus fire exclusively during waking and support many physiological functions. We investigated the role of the endovanilloid N-oleoyldopamine (OLDA) in TMN, where dopamine synthesis and its conjugation with oleic acid likely occur. We show that several known targets of OLDA including TRPV1 and cannabinoid receptors are expressed in HA neurons. In contrast to capsaicin, which failed to increase firing of HA neurons in TRPV1 knockout mice (TRPVI KO), OLDA was still able to induce excitation. This excitation was not sensitive to the blockade of cannabinoid receptors 1 and 2 and could result from OLDA interaction with GPR119, as the ligand of GPR119, oleoylethanolamide (OEA), also increased the firing of HA neurons. However, we ruled out this possibility as OEA- (but not OLDA-) excitation was abolished by the PPAR (peroxisome proliferator activated receptor) alpha antagonist MK886. The dopamine uptake blocker nomifensine blanked OLDA-excitation and dopamine receptor antagonists abolished the OLDA action in TRPV1 KO mice. Therefore OLDA excites HA neurons through multiple targets suggesting a central role of the histaminergic system in the behavioral stimulation seen after systemic OLDA application.
Asunto(s)
Dopamina/análogos & derivados , Histamina/metabolismo , Área Hipotalámica Lateral/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotransmisores/farmacología , Animales , Dopamina/farmacología , Área Hipotalámica Lateral/citología , Área Hipotalámica Lateral/crecimiento & desarrollo , Área Hipotalámica Lateral/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/citología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Metabotropic glutamate receptor 5 (mGluR5) is a drug target for central nervous system disorders such as fragile X syndrome that involve excessive glutamate-induced excitation. We tested the efficacy of a novel negative allosteric modulator of mGluR5 developed by Merz Pharmaceuticals, MRZ-8456, in comparison to MPEP and AFQ-056 (Novartis, a.k.a. mavoglurant) in both in vivo and in vitro assays in a mouse model of fragile X syndrome, Fmr1KO mice. The in vivo assays included susceptibility to audiogenic-induced seizures and pharmacokinetic measurements of drug availability. The in vitro assays included dose response assessments of biomarker expression and dendritic spine length and density in cultured primary neurons. Both MRZ-8456 and AFQ-056 attenuated wild running and audiogenic-induced seizures in Fmr1KO mice with similar pharmacokinetic profiles. Both drugs significantly reduced dendritic expression of amyloid-beta protein precursor (APP) and rescued the ratio of mature to immature dendritic spines. These findings demonstrate that MRZ-8456, a drug being developed for the treatment of motor complications of L-DOPA in Parkinson's disease and which completed a phase I clinical trial, is effective in attenuating both well-established (seizures and dendritic spine maturity) and exploratory biomarker (APP expression) phenotypes in a mouse model of fragile X syndrome.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/uso terapéutico , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Indoles/uso terapéutico , Isoquinolinas/uso terapéutico , Fenotipo , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/química , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Indoles/química , Indoles/farmacología , Isoquinolinas/química , Isoquinolinas/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Pirazoles/química , Pirazoles/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Distribución AleatoriaRESUMEN
BACKGROUND: Cerebral evoked potentials (CEP) induced by colorectal distension (CRD) in conscious rats provides a novel method in studies of visceral sensitivity. The aim of this study was to explore the pharmacological effect on CEP of compounds known to reduce the visceromotor response to CRD. METHODS: Epidural electrodes were chronically implanted in eight female Sprague-Dawley rats. Evoked potentials were elicited by colorectal rapid balloon distensions (100â¯ms, 80â¯mmHg) and the effect of WIN55 (cannabinoid CB receptor agonist), clonidine (adrenergic α2 receptor agonist), MPEP (mGluR5 receptor antagonist), pregabalin (ligand of α2δ subunits in voltage-gated calcium channels) and baclofen (GABA-B receptor agonist) on amplitudes and latency of CEP were determined. RESULTS: WIN55 (0.1⯵molâ¯kg-1), clonidine (0.05⯵molâ¯kg-1), MPEP (10⯵molâ¯kg-1) and pregabalin (200⯵molâ¯kg-1) caused a significant, pâ¯<â¯0.05, reduction of the N2 to P2 peak-to-peak amplitude by 23⯱â¯8%, 25⯱â¯8%, 39⯱â¯5%, and 47⯱â¯6% respectively. Baclofen (9⯵molâ¯kg-1) induced a prolongation of the N2 peak latency of 18⯱â¯4% but had no significant effect on the amplitudes. CONCLUSION: The obtained results suggest that MPEP, WIN55, clonidine, and pregabalin reduce visceral nociceptive input to the brain, whereas the lack of effect of baclofen on CRD evoked CEP amplitudes suggest that the effect on VMR is not due to a direct analgesic effect. Brain responses to colorectal distension provide a useful tool to evaluate pharmacological effects in rats and may serve as a valuable preclinical model for understanding pharmacological mechanisms related to visceral sensitivity.
Asunto(s)
Baclofeno/farmacología , Benzoxazinas/farmacología , Corteza Cerebral/fisiología , Clonidina/farmacología , Colon/efectos de los fármacos , Dilatación Patológica/fisiopatología , Potenciales Evocados/fisiología , Morfolinas/farmacología , Naftalenos/farmacología , Pregabalina/farmacología , Piridinas/farmacología , Animales , Femenino , RatasRESUMEN
2-Methyl-6-(phenylethynyl)pyridine (MPEP), a negative allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, protects hepatocytes from ischemic injury. In astrocytes and microglia, MPEP depletes ATP. These findings seem to be self-contradictory, since ATP depletion is a fundamental stressor in ischemia. This study attempted to reconstruct the mechanism of MPEP-mediated ATP depletion and the consequences of ATP depletion on protection against ischemic injury. We compared the effects of MPEP and other mGluR5 negative modulators on ATP concentration when measured in rat hepatocytes and acellular solutions. We also evaluated the effects of mGluR5 blockade on viability in rat hepatocytes exposed to hypoxia. Furthermore, we studied the effects of MPEP treatment on mouse livers subjected to cold ischemia and warm ischemia reperfusion. We found that MPEP and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) deplete ATP in hepatocytes and acellular solutions, unlike fenobam. This finding suggests that mGluR5s may not be involved, contrary to previous reports. MPEP, as well as MTEP and fenobam, improved hypoxic hepatocyte viability, suggesting that protection against ischemic injury is independent of ATP depletion. Significantly, MPEP protected mouse livers in two different ex vivo models of ischemia reperfusion injury, suggesting its possible protective deployment in the treatment of hepatic inflammatory conditions.